Expression of the glucocorticoid receptor in the human adrenal cortex.

Glucocorticoids produced in the adrenal cortex act by binding to a specific intracellular protein, the glucocorticoid receptor (GR), which then modulates gene transcription in target tissues. Whether the adrenal cortex itself is a glucocorticoid target tissue has not been analyzed as yet. Since the presence of GR would be a prerequisite for such "intracortical" glucocorticoid action, this study was designed to analyze GR expression in the normal human adrenal gland using RT-PCR, Western blot, and immunohistochemistry. RT-PCR revealed the presence of GR mRNA in adrenal cortex as well as in NCIh295 cells. These results were confirmed at the protein level by Western blot employing a specific anti-human GR antibody. Immunohistochemically, weak GR staining was observed in the adrenal medulla. In contrast, GR was strongly expressed in the adrenal cortex with the zona reticularis showing the most intense staining. Transfection of a GR-responsive luciferase reporter gene into NCIh295 cells resulted in dexamethasone-dependent induction of luciferase activity, indicating that GR is functional in this tissue. In this study, we show for the first time that GR is expressed in the human adrenal cortex. Its preferential expression in the zona reticularis may indicate a functional role in the regulation of adrenal androgen biosynthesis.

Presence of a 5-HT7 receptor positively coupled to adenylate cyclase activation in human granulosa-lutein cells.

Although serotonin (5-HT) has been shown to stimulate progesterone production by human granulosa-lutein cells (hGLC), the receptor type and associated signaling pathway remain uncharacterized. We report here that 5-HT receptors in these cells are positively coupled to adenylate cyclase activity. Formation of cAMP was stimulated by 5-HT and its agonists in a dose- and time-dependent manner. Mianserin, amoxapine, and loxapine were equipotent in antagonizing 5-HT-induced cAMP formation. For both cAMP formation in cells and adenylate cyclase assay using membrane fractions, the rank order of potency for agonists of 5-HT were: 5-carboxy-aminotryptamine >5-HT> or =5-methoxytryptamine, consistent with a typical pharmacological profile of human 5-ht7 (h5-ht7) receptor. Sequence data of amplified complementary DNA fragments reverse transcribed from hGLC RNA revealed complete identity with published sequence of h5-ht7 receptor complementary DNA. Northern analysis showed the presence of 2.8-kb h5-ht7 transcripts in hGLC. The three variants h5-ht7A, h5-ht7B, and h5-ht7D were also detected in hGLC. Preincubation of hGLC with 5-HT (10(-8)-10(-6) M) resulted in a marked reduction in the cAMP response when the cells were subsequently stimulated with gonadotropin, and this heterologous desensitization could be reversed by 5-ht7 receptor antagonist clozapine. These data demonstrate that h5-ht7 receptor is present and stimulate cAMP formation in hGLC. In addition, the h5-ht7 receptor seems to be implicated in the heterologous down-regulation hCG-stimulated cAMP response in hGLC, with a possible ramification for luteal insufficiency.

Natriuretic peptides in the human testis: evidence for a potential role of C-type natriuretic peptide in Leydig cells.

Functional studies indicate that natriuretic peptides have direct effects on Leydig cells of the testis. In this report, we demonstrate local synthesis of one member of the natriuretic peptide family, C-type natriuretic peptide (CNP), in Leydig cells of human testes. Using RT-PCR assays, messenger RNA (mRNA) for the CNP precursor was detected in human testis and found to be prominently expressed in Leydig cells. Immunohistochemical analyses revealed CNP to be almost exclusively associated with Leydig cells. Distinct differences in the staining intensity-including cells without detectable staining-suggest a heterogeneity of CNP expression within the Leydig cells. Moreover, the presence of transcripts for the CNP receptor, a particulate guanylate cyclase, termed GC-B, was demonstrated by RT-PCR in human testis and in isolated Leydig cells. The expression of this receptor in human testis membranes could be confirmed by affinity labeling with 125I-labeled CNP. These findings demonstrate, for the first time, the production of a natriuretic peptide in human Leydig cells. The occurrence of CNP and its receptor in the human testis points to a local role of the peptide, presumably acting in an auto- or paracrine manner to modulate organ-specific functions.

The expression of the RLF/INSL3 gene is reduced in Leydig cells of the aging rat testis.

The relaxin-like factor (RLF), which is the product of the INSL3 gene, is highly expressed in the fetal and adult-type Leydig cells of all species so far examined. In adult testes it is upregulated at puberty but appears subsequently to be expressed in a constitutive manner, independently of acute changes in the hypothalamic-pituitary-gonadal (HPG) axis. Functional hypogonadism with decreased testosterone is prevalent in the aging male. In order to test whether this is a property of the HPG axis, or of the Leydig cells themselves, RLF/INSL3 was used as an independent marker to assess rat Leydig cell differentiation status. Hybridization analysis showed that in the testes of old (2 years) rats, RLF/INSL3 mRNA expression was dramatically reduced, compared to young (3 months) animals. This was also evident at the protein level using immunohistochemistry. The results suggest that increasing functional hypogonadism in older male mammals is likely caused by a dedifferentiation of the Leydig cells themselves.

Synthesis of C-type natriuretic peptide (CNP) by immortalized LHRH cells.

Former studies have indicated an influence of natriuretic peptides on LHRH secretion. In this report we demonstrate local synthesis of CNP in immortalized LHRH neurons (GT1-7 cells). Using reverse transcription-polymerase chain reaction and RNase protection assays a transcript for the CNP precursor was identified in these cells. Immunocytochemical data revealed the presence of the peptide CNP in GT1 cells, using a specific polyclonal antiserum against CNP. Electron microscopic immunohistochemical investigations also showed the strongest CNP-immunoreactivity in some small vesicles, providing initial evidence for the potential secretion of this peptide by immortalized LHRH neurons. Subsequent experiments demonstrated also that CNP elevates LHRH production in static cultures of GT1 cells. These data show for the first time the co-production of the functionally relevant natriuretic peptide, CNP, by immortalized LHRH neurons. Together with the recent demonstration of CNP receptor expression by these cells, we suggest that CNP may represent a novel autocrine regulator of LHRH neuronal activity. It remains to be elucidated, however, to what extent CNP expression in immortalized LHRH neurons reflects a co-localization in situ of CNP and LHRH peptides.

Cloning of a cDNA encoding a novel putative G-protein-coupled receptor expressed in specific rat brain regions.

A cDNA clone encoding a novel putative G-protein-coupled receptor was isolated from a rat brain cDNA library using a PCR-amplified cDNA fragment as a hybridization probe. The 3,615-bp-long nucleotide sequence predicts a single open reading frame of 1,173 bp coding for 391 amino acids, giving a calculated molecular weight of 42.75 kD. The amino acid sequence shares features common to many other receptors, including the seven membrane-spanning hydrophobic regions and putative asparagine-linked glycosylation and phosphorylation sites. Northern blot analysis reveals that a corresponding approximately 3.7-kb mRNA is expressed in specific brain regions such as hypothalamus, cortex, hippocampus, and thalamus but not in other organs analyzed. Although the ligand for this receptor has not yet been identified, it shares some similarities with the vascular type-1 angiotensin II receptor, the vasoactive intestinal peptide (VIP) receptor, and the chemotactic receptors for human C5a anaphylatoxin and the formyl peptide fMet-Leu-Phe.

Measurement of fatty acid oxidation in premature newborn infants with the 13C-triolein breath test.

The 13C-triolein breath test is a method giving evidence of extent and rate of fatty acid oxidation in newborn infants on parenteral nutrition. The test has the special advantage of being non-invasive. Triolein labeled with the stable carbon isotope 13C and emulsified in soybean-oil is used as a tracer. 10 mg of 13C triolein per kg body weight are administered intravenously. The 13CO2 resulting from the fatty acid oxidation is analysed in expired breath by ratio-mass-spectrometry. The calculated 13C elimination is representative of the rate of fatty acid oxidation during the examination period. First studies on 15 premature infants have shown that an average of 27.0 +/- 1.8% of the dose administered is oxidized within 4 h. The present results suggest that the oxidation rate may be related to the maturity of the prematurely born infants.

Endothelin and endothelin receptors A and B in the human testis.

Human testicular capillaries interconnect Leydig cells and seminiferous tubules. Microcirculation and blood flow are therefore essential for the maintenance of spermatogenesis. The expression and the localisation of ET (endothelin) and its receptors in testicular tissue, in seminiferous tubules and in human testicular capillaries were studied. ET-1 mRNA was detected in whole testicular tissue and in seminiferous tubules whereas isolated testicular capillaries were negative. Big ET-1 (Big endothelin 1) and ET peptides were localised in Leydig and Sertoli cells whereas interstitial and intramural capillaries (within the lamina propria) remained unstained. ET was also found in mature spermatids. ET-A (endothelin receptor A) mRNA was detected in seminiferous tubules and whole testicular tissue whereas testicular blood vessels were negative. ET-A immunostaining was displayed in Leydig and Sertoli cells and in spermatids. ET-B (endothelin receptor B) mRNA was detected in whole testicular tissue, seminiferous tubules and in testicular capillaries. ET-B peptide was prominent in Leydig cells, peritubular cells, endothelial cells and pericytes of interstitial and intramural capillaries as well as in vascular endothelial and smooth muscle cells. From these results we conclude that ET produced in Leydig and Sertoli cells can act in a paracrine manner via ET-B on the human testicular microvasculature and the peritubular cells. The presence of both ET-A and ET-B in Leydig cells and of ET-A in Sertoli cells leads to the assumption that ET could influence these cells as an autocrine factor.

Lipid infusion in premature infants suffering from sepsis.

In as much as possible side effects attributing to insufficient fat clearance with hyperlipemia, parenteral lipid administration to septic premature infants is controversial. In this study serum triglyceride and free fatty acid concentrations of nine low birth weight infants with septicemia and 21 low birth weight infants without septicemia were measured. Acidosis, hypoxia, hyperglycemia, and cardiovascular insufficiency were treated before parenteral lipid infusion was started. There was no occurrence of septic shock. In the course of fat infusion with 3 g/kg body weight per day in low birth weight infants without systemic infection we only found triglyceride concentrations of 1.15 mmol/liter and free fatty acid levels of 1.05 mmol/liter. Premature infants with septicemia showed, under fat application of 2 g/kg body weight per day, mean triglyceride levels of 1.67 mmol/liter and free fatty acid values of 1.94 mmol/liter. The highest concentrations occurred at 3 g fat/kg body weight per day with triglycerides of 2.02 mmol/liter and free fatty acids of 2.06 mmol/liter. They indicate a reduced clearance and support earlier findings of reduced utilization of infused fat in premature infants with septicemia. Triglyceride concentrations more than 1.7 mmol/liter probably induce an increase of phagocytosis of the fat particles with the effect of a partial block of the reticuloendothelial system and an impairment of pulmonary diffusion capacity. Therefore, we suggest dosages no higher than 2 g fat/kg body weight per day to low birth weight infants and we advise to check the triglycerides daily. Hypertriglyceridemia implicates an immediate reduction or total interruption of the lipid infusion until normal triglyceride values are regained.

Impaired fat utilization in parenterally fed low-birth-weight infants suffering from sepsis.

Lipid infusion in low-birth-weight infants suffering from sepsis is still controversial. Consequently, we investigated the fat tolerance in six low-birth-weight infants with sepsis and 15 low-birth-weight infants without sepsis. For measurement of fat clearance, we assayed the serum concentrations of triglycerides enzymatically, and of the free fatty acids by colorimetric micromethod. The fatty acid oxidation was analyzed with the [13C]triolein breath test by means of ratio-mass spectrometry. The infants were maintained on continuous parenteral nutrition with various amounts of soybean oil emulsion (1 g, 2 g, and 3 g fat/kg body weight per day). Comparing the lipid infusion of 1 and 2 g fat/kg body weight per day between the two groups, we found triglyceride and free fatty acid values in both groups to be in the normal range. At a dose of 3 g of fat/kg body weight per day, septic low-birth-weight infants showed a significantly higher concentration of triglycerides (2.02 +/- 0.46 mmol/liter) and of free fatty acids (2.06 +/- 0.45 mmol/liter) than the nonseptic low-birth-weight infants (triglycerides: 1.09 +/- 0.43 mmol/liter; free fatty acids: 1.05 +/- 0.41 mmol/liter). The low-birth-weight infants with sepsis showed a reduced fat oxidation rate of 16.0 +/- 1.5% in contrast to that of the low-birth-weight infants without sepsis, whose rate was 38.4 +/- 1.8%. Accordingly, we apply dosages not exceeding 2 g of fat/kg body weight per day to septic low-birth-weight infants.

Fat elimination in parenterally fed low birth weight infants during the first two weeks of life.

Eighteen low birth weight infants (27-34 wk gestation) were given supplementary parenteral nutrition via peripheral veins of a maximal dose of 8.5 g glucose, 2.5 g amino acids (Aminovenös päd 10%) and 2 g soybean oil egg lecithin emulsion (Intralipid 10%) kg body weight/24 hr. The fat emulsion was infused continuously at a rate of 0.084 g/kg body weight/hr. The elimination of Intralipid from the blood stream was controlled by enzymatic determination of serum triglyceride concentrations, and the fatty acid pattern of the serum lipids was determined by gas chromatography. The serum triglyceride concentrations were 0.60 +/- 0.16 mmol/liter on the 1st day, increased to 0.96 +/- 0.29 mmol/liter up to the 5th day, and approached a level around 0.90 mmol/liter in the further course. No hypertriglyceridemia was noted. The fatty acid pattern of the serum lipids showed a linoleic acid fraction of 8.1 +/- 4.0% in the beginning, which was followed by a continuous increase up to 27.8 +/- 4.8% on the 7th day. No significant changes were noticed thereafter. The levels were within the normal limits as found in 2-wk-old enterally fed preterm infants of comparative maturity (25.6 +/- 3.4%). We conclude that the preterm infants can eliminate Intralipid from the blood stream if maximal dosage and infusion rate, as described above, are applied.

New aspects of Leydig cell function.

Previous studies indicated that the Leydig cells of the human testes show similarities to neuroendocrine cells. In this context, the local synthesis of two neuroactive signaling molecules, namely nitric oxide (NO) and C-type natriuretic peptide (CNP), both acting via the second messenger, cyclic guanosine monophosphate (cGMP), might be of physiological relevance. By immunoblotting, immunohistochemical analyses and affinity crosslinking experiments, respectively, the presence of soluble guanylate cyclase (sGC), the NO receptor, and of guanylate cyclase B (GC-B), representing the CNP receptor, was demonstrated in Leydig cells, seminiferous tubules and blood vessels of the human testis. Moreover, cGMP and its binding protein cGMP-dependent protein kinase type I (GK I) were found in these structures. The functional activity of the two receptors was proved by generation of cGMP in response to treatments with the NO donor, sodium nitroprusside (SNP), and with CNP, respectively. As indicated by immunohistochemical analyses and by treatments of cells with either SNP or CNP, human Leydig tumour cells and MA10 cells, representing a mouse Leydig tumour cell line, were found to be distinguished by a reduced expression of the receptors for NO and CNP. Furthermore, expression levels of the components of the two cGMP-generating systems were found to be widely unchanged in Leydig cells during different ontogenetic stages. Though cGMP has been shown to influence testosterone release, the constant developmental expression patterns of NO and CNP apparently independent of differences in androgen production, the down-regulation of their receptors in tumorous cells, and the presence of GK I, may point to additional autocrine functions of these factors and of cGMP in Leydig cells. Moreover, possible paracrine actions of NO and CNP may include relaxation of seminiferous tubules and blood vessels in order to modulate sperm transport and testicular blood flow, respectively. These findings suggest that Leydig cell-derived factors may exert activities different from or in addition to those involved in the regulation of testosterone production.

[Seizures and hyponatremia in a newborn infant]. / Krampfanfälle und Hyponatriämie bei einem Neugeborenen.

A three days old mature female newborn presented with seizures. The diagnostic data revealed a hyponatraemia (113 mmol/l) as pathogenic origin. Further causes of convulsions could be excluded. Administering saline solution (0.9%) intravenously the plasma sodium level reached normal ranges within 36 hours. We propose that an excessive enteral administration of glucose solution during the first three days of life must have led to dilutional hyponatraemia. This suggestion is in accordance with observations in older children presenting with similar symptoms.

[Group B streptococci: the most common cause of neonatal septicemia (author's transl)]. / B-Streptokokken als häufigste Erreger der Neugeborenensepsis. Einleitung, Häufigkeit, Klinik, Therapie.

Between 1965--78 118 newborns with septicemia have been treated in the Children's Hospital of the Free University Berlin. Microorganisms identified were streptococci in 32 cases, 27 of which were group B streptococci (increasing number since 1973). In 1978 group B streptococci were responsible for 44% of all the septicemia cases as well as for 12% of all newborn deaths. The incidence of group B streptococcal septicemia in newborn babies is 1/1000 live births for the Berlin region. 2 patients presented the late-onset type of group B streptococcal neonatal sepsis; both survived having neurological sequelae. 25 newborns belonged to the early-onset group, the mortality rate in this group is 56%. The clinical features, bacteriological findings and risk factors are summarized in table form. There could be an influence related to the maternal blood type. Histological examinations in 5 placentae revealed signs of amniotic infection.

Plasma amino acids in supplementary parenteral nutrition of preterm infants. Effect of different quantities of amino acid infusion and comparison with enteral feeding.

The present investigation aims to determine quantity and quality of the amino acid (AA) solution to be used in supplementary parenteral nutrition (SPN). We established the plasma AA concentrations of preterm infants (birthweight 1160-1940 g, mean 1540; gestational age 29-30 weeks, mean 32) divided into three groups. Group I (n = 11) and group II (n = 12) were put on a standardised SPN regimen starting with an intravenous supply of 2.5 resp. 1.5 g AA/kg/day. Infants of group III were formula-fed, and served as controls. A total of 231 aminograms was obtained during the first two weeks of age. Comparison of groups I and II to group III revealed plasma accumulation of six AA in group I. Supplementation in group II resulted in a normal pattern, except alanine, proline, and methionine. However, only deviations of proline and methionine may be judged as imbalances, and lowering in composition may be considered. We conclude that the low intravenous AA intake employed in group II may be preferred in SPN of preterm infants.

[Current status of parenteral feeding with fat infusions. Clinical experiences with premature and newborn infants]. / Aktueller Stand der parenteralen Ernährung mit Fettinfusionen. Klinische Erfahrungen bei Früh- und Neugeborenen.

By administration of fat emulsion a well-balanced parenteral nutrition concerning calories is possible in newborn infants. Investigations with great amounts of fat during and after a short-time infusion have shown that the maximal fat clearance of very-low-birth-weight and small-for-gestational-age newborn infants is limited. Heparin can improve the lipid clearance and reduce hyperlipaemia occurring under lipid application. The fat oxidation is not affected by heparinization. Extent and velocity of the fatty acid utilization can be judged by the 13C-triolein breath test. Under the clinical conditions of a continuous long-term infusion with 2 g fat/kg BW/day a complete fat clearance is observed in low-birth-weight infants with respiratory distress syndrome and septicaemia and small-for-gestational-age newborn infants. The determination of serum triglycerides is considered to be a sufficient control of fat clearance in respect to clinical concerns. The fat emulsion is continuously applied for 24 h and is being increased stepwise up to a dosage of 2-3 g/kg BW/day. Before starting parenteral nutrition, acidosis, hypoxaemia, hyperglycaemia and insufficient circulation must have been treated. Contraindications to this are shock, disturbances of blood coagulation and of fat metabolism. Complications are avoided by using an adapted and standardized nutritional programme under sufficient clinical and laboratory control.